ORIGINAL ARTICLE EXPERIMENTAL/SPECIAL TOPICS

Processing Technique for Lipofilling Influences Adipose-DerivedStem Cell Concentration and Cell Viability in Lipoaspirate

Miles Pfaff • Wei Wu • Elizabeth Zellner •

Derek M. Steinbacher

Received: 14 September 2013 / Accepted: 9 December 2013 / Published online: 8 January 2014

� Springer Science+Business Media New York and International Society of Aesthetic Plastic Surgery 2014

Abstract

Background Autologous fat grafting is a highly used

technique in plastic and reconstructive surgery. Several fat-

processing techniques have been described, with centrifu-

gation frequently touted as the optimal method. Processing

is one factor important for maximizing cell viability and

adipose-derived mesenchymal stem cell (ADSC) concen-

trations. This study compared two methods of fat prepa-

ration (centrifugation vs Telfa-rolling) to determine which

technique results in the greatest degree of cell viability and

ADSC concentration.

Methods Abdominal fat was harvested from five patients.

Equal aliquots were divided and processed by both cen-

trifugation and Telfa-rolling. Samples were analyzed for

ADSC proportions via flow cytometry and cell viability

using methylene blue-based cell counting. Paired t tests

were performed on all samples, and a P value lower than

0.05 was considered statistically significant.

Results Telfa-rolling processing resulted in a higher per-

centage of isolated ADSCs (P \ 0.5 for 3 of 4 parameters)

and a significantly higher number of viable cells (P \ 0.05).

Conclusion Telfa-rolling results in a higher proportion of

ADSCs and greater cell viability than centrifugation for

donor adipose graft preparation. Further studies are nec-

essary to confirm whether optimal preparation translates to

improved augmentation and cell take at the recipient site.

Level of Evidence IV This journal requires that authors

assign a level of evidence to each article. For a full

description of these Evidence-Based Medicine ratings,

please refer to the Table of Contents or the online

Instructions to Authors www.springer.com/00266.

Keywords Telfa-rolling � Centrifugation � Fat grafting �Fat processing � Lipoaspirate � Lipoaspiration � Adipose-

derived mesenchymal stem cells Adipose-derived stem

cells � ADSC � Lipofilling

Autologous fat grafting is exceedingly popular, with broad

applicability in plastic and reconstructive surgery. Fat

grafting can impart contours and augmentation, nourish

tissue, modulate scar tissue, and exhibit regeneration at the

recipient site [1–5]. Factors taken into consideration when

fat grafting is performed include donor-site location, method

of harvest (manual vs machine-assisted liposuction), prep-

aration and injection technique, and recipient-site suitabil-

ity. Multiple techniques at all stages of the process have been

described and tested to establish a standardized, optimal

approach.

Adipose-derived mesenchymal stem cells (ADSC) are

an accessible stem cell source within lipoaspirate [6], and

accumulating evidence suggests that fat graft survival is

directly related to cell viability and may be enhanced by

increased ADSC concentrations [7].

A number of techniques have been developed and tested

to achieve the highest concentration and quality of adipo-

cytes from lipoaspirate. Centrifugation, a common method

of fat graft processing, facilitates clearance of potentially

deleterious materials (red blood cells and oil) from the

tissue sample and permits large-volume graft processing. It

is posited, however, that the process of centrifugation may

negatively influence graft survival [8, 9].

M. Pfaff � W. Wu � E. Zellner � D. M. Steinbacher (&)

Section of Plastic and Reconstructive Surgery, Yale University

School of Medicine, 3rd Floor, Boardman Building,

330 Cedar Street, New Haven, CT 06520, USA

e-mail: derek.steinbacher@yale.edu; derek.steinbacher@gmail.com

123

Aesth Plast Surg (2014) 38:224–229

DOI 10.1007/s00266-013-0261-7

Telfa-rolling [10], a filter-based technique that entails

spreading or rolling lipoaspirate on absorbent, nonadherent

gauze, permits simple, efficient, and atraumatic processing

without the use of equipment and may be ideal for smaller-

scale procedures.

To date, it is unclear which processing method enables

enrichment of ADSCs and fat cells from harvested tissue.

This study aimed to compare the levels of both cell via-

bility and ADSC population proportions using two differ-

ent methods of fat graft processing: centrifugation and

Telfa-rolling processing.

Materials and Methods

Patients, Fat Graft Harvesting, and Lipoaspirate

Processing

Consent was obtained from all donors before fat harvest-

ing, as indicated by the human investigation committee

(Protocol #1204010149). For fat harvesting, 10 mL of 1 %

lidocaine (with 1:100,000 epinephrine) was administered to

the donor site (on each side of the abdomen), and the lip-

oaspirate was harvested from each patient using a 10-mL

syringe under manual suction, as described previously [11].

Lipoaspiration was performed on both sides of the

abdomen to ensure optimal quality of harvested fat. For

centrifuge processing, 1 mL of lipoaspirate was placed into

a 10-mL syringe with sterile phosphate-buffered saline

(PBS) to achieve 10 mL of total volume and centrifuged at

1,500 revolutions per minute (rpm) for 3 min. The enriched

fat (supernatant) then was collected for further processing

(Fig. 1a).

For Telfa-rolling processing, the lipoaspirate was placed

on Telfa gauze pads (product used in this study, Kendall

Co., Mansfield, MA, USA) and gently rolled or spread back

and forth until it was determined that oil and blood had

been sufficiently removed (Fig. 1b) for approximately 30 s.

Then, 1 mL of processed lipoaspirate was placed in a

10-mL syringe with PBS to achieve 10 mL of total volume

for further analysis.

Cell Isolation and ADSC Population and Cell Viability

Quantification

Cells were isolated and cultured as described previously

[11]. All comparisons were made from cells cultured

before the first passage. For 60 min, 1 mL of processed fat

was digested with Dulbecco modified Eagle medium

(DMEM)-low glucose (Gibco, Grand Island, NY, USA)

containing 0.15 % collagenase I (Worthington Biochemi-

cal, Lakewood, NJ, USA) at 37 �C. Stromal vascular

fraction, the stem-cell-enriched fraction of lipoaspirate,

was collected by centrifugation, filtered through a 40-lm

strainer, and suspended in complete medium consisting of

DMEM-low glucose, 10 % fetal bovine serum (FBS)

Fig. 1 Centrifugation and Telfa-rolling techniques. a Syringes

(10 mL) are placed in a centrifuge (arrows) and spun to facilitate

clearance of potential contaminants such as blood, crystalloid, and oil

(bottom panel). b For Telfa-rolling, the lipoaspirate is placed on

nonadhesive Telfa gauze (top panel) and rolled or spread back and

forth to remove potential contaminants

Aesth Plast Surg (2014) 38:224–229 225

123

(Lonza, Allendale, NJ, USA), and 1 % penicillin–strepto-

mycin (Gibco).

The ADSC population proportions were quantified using

flow cytometry [11]. Briefly, 1 9 106 nucleated cells were

resuspended with cold fluorescence-activated cell-sorting

(FACS) buffer (PBS containing 2 % FBS) in FACS tubes.

The cells were rinsed with chilled FACS buffer and incu-

bated with all-specific conjugated primary antibody and the

corresponding isotype control.

The treated cells were rinsed with chilled FACS buffer

and analyzed in triplets using an FACS LSR-II device (BD

Biosciences, San Jose, CA, USA). The following human

antibodies were used: CD44 (fluorescein isothiocyanate),

CD73 (phycoerythrin), CD90 (alkaline phosphatase), and

CD105 (alkaline phosphatase) (BD Biosciences).

Antibody specificity is as follows: CD44 is specific to

phagocytic glycoprotein-1 and expressed on cells during

hematopoiesis and lymphocyte activation; CD73 is specific

to ecto-50 nucleotidase and expressed on mesenchymal

stem cells; CD90, a protein expressed by a small subset of

human fetal liver cells, cord blood cells, and bone marrow

cells, is believed to be useful for identifying high prolif-

erative potential colony-forming cells; and CD105 is an

integral membrane homodimer protein expressed on mes-

enchymal stem cells.

Isolated cells from individual donors were mixed with

3 % acetic acid using methylene blue (STEMCELL Tech-

nology; Vancouver, Canada), and the number of nonstained

nucleated cells was quantified under phase-contrast

microscopy (Carl Zeiss, Oberkochen, Germany).

Statistical Analysis

Statistical analysis was performed using Microsoft Excel,

version 14.0.0 (Microsoft Office 2011; Microsoft; Red-

mond, WA, USA) and SPSS Statistics, version 19 (IBM,

Armonk, NY, USA). The mean value was calculated and

reported with the standard deviation. A paired t test was

performed to compare cell viability counts and ADSC

population proportions. An observed P value of 0.05 was

considered statistically significant.

Results

Lipoaspirate was harvested from the lower abdomen via an

umbilical access point of five patients: a 12-year-old boy, a

17-year-old girl, a 27-year-old woman, a 64-year-old man,

and a 68-year-old woman (mean age, 37.6 ± 23.7 years).

No comorbidities were noted in the patient cohort.

Flow cytometry showed significantly higher levels of

ADSC in Telfa-processed lipoaspirate than in centrifuga-

tion-processed lipoaspirate (Fig. 2). The mean percentage of

cells expressing the combination CD73 and CD105 was 3.1

in the Telfa group and 4.3 in the centrifugation group

(P = 0.64). The cells expressing CD73 and CD44 were 2.3

in the centrifugation group and 6.5 in the Telfa group

(P = 0.0). Cells expressing CD73 and CD90 were 2.0 in the

centrifugation group and 5.7 in the Telfa group (P = 0.03).

Finally, the mean percentage of cells expressing the com-

bination CD90 and CD44 was 2.8 % in the centrifugation

group and 7.5 in the Telfa group (P = 0.05).

In a separate experiment, Telfa-processed liposapirate

resulted in an more viabile cells than centrifugation-pro-

cessed lipoaspirate (1.7 vs 1.3 [9106 cells]) (P = 0.25),

respectively (Fig. 3).

Discussion

Autologous fat grafting has myriad applications in plastic

and reconstructive surgery. The utility of this technique,

however, is limited by unpredictable resorption rates,

which reach 55 % [12–14]. Thus, much of the research has

Fig. 2 Flow cytometry for quantification of adipose-derived mesen-

chymal stem cell (ADSC) population proportions in centrifugation-

and Telfa-processed lipoaspirate. Mean ± standard error of mean.

*P \ 0.05

Fig. 3 Cell viability in centrifugation- and Telfa-processed lipoaspi-

rate. Mean ± standard error of mean. *P = 0.003

226 Aesth Plast Surg (2014) 38:224–229

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focused on both development and refinement of harvesting

and processing techniques to improve fat graft viability,

with increasing emphasis placed on the importance of

ADSC.

Findings show that ADSCs are a widely accessible

population of precursor stem cells that exhibit trilineage

(adipogenic, chondrogenic, and osteogenic) differentiation

capacity [6]. The potential benefits of using ADSCs as an

adjunct therapy to improve fat graft survival have been

investigated previously. Studies have shown that supple-

mentation of fat grafts with ADSCs prolongs graft survival

and maintains fat graft viability (absence of necrosis or

fibrosis and increased vascularization) in animal fat graft

models [15–19].

Clinically, Yoshimura et al. [20] have shown cell-assisted

lipotransfer-mediated fat grafting (fat grafts enriched with

ADSCs and other cell populations) can be used to treat

facial lipoatrophy as well as breast augmentation [21] and

revision [13]. Furthermore, in a recent randomized con-

trolled trial by Kolle et al. [7], lipofilling with ex vivo

expanded ADSCs resulted in longer fat graft volume sur-

vival, providing supportive evidence that ADSC enrichment

may provide a benefit over traditional nonenriched fat grafts.

Although the mechanism of ADSC-mediated fat graft

survival has not been elucidated to date, it is posited that

mesenchymal stem cells provide a conducive environment

for graft vascularization and thus survival, primarily via

hypoxia-induced vascular endothelial growth factor

(VEGF)-mediated angiogenesis. Lu et al. [16] found that

fat graft survival was enhanced by ADSC enrichment and

further potentiated by VEGF supplementation. Similar

results were observed after enrichment of bone marrow–

derived mesenchymal stem cell–enriched fat grafts trans-

fected with VEGF [15].

Accumulating evidence suggests that ADSC concentra-

tions and cell viability can be influenced at all stages of the

fat-grafting process, from donor and donor-site selection to

graft placement. Multiple studies have examined anatomic

site-specific benefits from improvement of graft survival,

concluding that no donor site is superior in terms of graft

viability [22–24]. That being said, site-specific differences

in ADSC quantity and concentrations of other cell popu-

lations may exist because findings have shown the lower

abdomen and, to a lesser extent, the inner thigh to be

ADSC-enriched sources of lipoaspirates [25]. However, a

recent study comparing stromal vascular fraction cell iso-

lates (a cellular mixture including stromal cells, vascular

endothelial cells, and ADSCs—the fraction isolated in this

study) from multiple donor sites showed no difference in

stromal vascular fraction cell numbers or graft survival

among donor sites [23]. In spite of this, the lower abdomen

was chosen as the donor site for all samples in the current

study.

Clinicians generally accept that the process of fat har-

vesting should be performed in an atraumatic fashion. This

assertion is supported by multiple studies demonstrating

preservation of cell viability and function using atraumatic

techniques such as manual syringe aspiration rather than

more aggressive techniques such as machine-assisted

liposuction [26–29]. However, a recent study comparing

manual- and machine-simulated pressures on fat grafts

showed no difference in graft viability in an animal fat

graft model [30]. When a setting requires large volumes of

fat grafts (e.g., breast and buttock augmentation) versus

smaller volumes (e.g. facial contouring), manual suction

may be impractical and thus machine-assisted lipoaspira-

tion often is used.

The fat-processing techniques previously described in

the literature include centrifugation, filtering, decantation,

and washing. The decision which technique to use often is

dependent on the volume of fat required for transplantation,

the availability of equipment (e.g., centrifuge), and the

surgeon’s own personal preference. Centrifugation is a

common method of processing, especially for larger-vol-

ume fat grafts, although Telfa-based processing may be

more ideal for smaller volumes.

Although the benefits of Telfa-based processing have yet

to be tested, multiple studies have compared centrifugation

with filtering techniques in an attempt to establish a superior

processing method [31, 32]. In one study, centrifugation-

based processing resulted in higher ADSC numbers but

decreased cell viability counts than decantation [33]. The

findings of the current study echo these results in showing

that centrifugation may disrupt cell viability compared with

Telfa-rolling, especially at higher revolutions per minute [8,

9]. Interestingly, the current study also found that Telfa-

rolling resulted in greater ADSC yields, a surprising finding

considering the ability afforded by centrifugation to achieve

increased cell concentrations within processed fat.

The technique of fat graft placement at the recipient site

is a critical determinant of graft survival. Findings have

shown that larger grafts, as required for breast and buttock

augmentation, are more likely to fail, presumably because

of limited diffusion capacity and low oxygen tension

within the graft. Khouri et al. [12] overcame this obstacle

by implementing the practice of external expansion for

breast augmentation before grafting to increase the graft–

recipient surface interface and to facilitate diffusion and

revascularization, resulting in better graft survival.

A recent report found that the shear stress generated

along the cannula during graft placement is inversely

related to graft survival [30]. Although small-volume

grafting may not necessitate such measures as external

expansion, administering smaller aliquots of fat under low

injection pressure, as the cited study suggests, may

improve fat graft survival in all scenarios.

Aesth Plast Surg (2014) 38:224–229 227

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The limitations to this study included the small sample

size and the absence of in vivo fat graft survival data.

Despite these limitations, the results, in addition to the

findings from previous laboratory and clinical studies,

suggest that Telfa-rolling processing of fat grafts may

provide a benefit over centrifugation. Laboratory-based

studies comparing the survival of fat grafts processed by

centrifugation or Telfa-rolling techniques are necessary to

provide a definitive answer.

Conclusion

This preliminary study demonstrated that Telfa-rolling fat

graft processing results in increased ADSC yields and cell

viability compared with centrifugation-based processing.

Telfa-rolling is an efficient and feasible preparation tech-

nique, particularly for lower-volume fat grafting, and may

provide ADSC-enriched samples.

Acknowledgments This study was supported by the Department of

Surgery, Yale University School of Medicine.

Disclosure All authors have no commercial associations or disclo-

sures that may pose or create a conflict of interest with the infor-

mation presented within this manuscript.

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